Wortmannin and Wortmannine Analogues from an Undescribed

3 days ago - In the course of our studies of coprophilous fungi as sources of antifungal agents, a strain of an undescribed species in the genus Niess...
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Wortmannin and Wortmannine Analogues from an Undescribed Niesslia sp. Nicole M. Dischler,† Lijian Xu,‡ Yan Li,‡ Connie B. Nichols,§ J. Andrew Alspaugh,§ Gerald F. Bills,‡ and James B. Gloer*,† †

Department of Chemistry, University of Iowa, Iowa City, Iowa 52242, United States Texas Therapeutic Institute, The Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at Houston, Houston, Texas 77054, United States § Departments of Biochemistry and Medicine, Duke University Medical Center, Durham, North Carolina 27710, United States

J. Nat. Prod. Downloaded from pubs.acs.org by UNIV OF TEXAS AT DALLAS on 03/08/19. For personal use only.

‡

S Supporting Information *

ABSTRACT: In the course of our studies of coprophilous fungi as sources of antifungal agents, a strain of an undescribed species in the genus Niesslia (TTI-0426) was isolated from horse dung collected in Texas. An extract from fermentation cultures of this strain afforded two new antifungal wortmannin derivatives, wortmannins C and D (1 and 2), as well as four additional new related compounds, wortmannines B1−B4 (3−6), containing an unusual ring system. The structures of these metabolites were established mainly by analysis of HRESIMS and 2D NMR data. Relative configurations were assigned using NOESY data, and the structure assignments were supported by NMR comparison with similar compounds. Wortmannins C and D showed activity against Cryptococcus neoformans and Candida albicans in disk assays, but low MIC potency observed for 1 was suggested to be due in part to efflux processes on the basis of assay results for a Schizosaccharomyces pombe efflux mutant in comparison to wild-type.

Cryptococcus species are among the most common causes of invasive fungal infections worldwide and are especially problematic for immunocompromised individuals such as those with HIV/AIDS.1,2 Current treatments still rely on a small number of antifungal agents discovered many years ago which remain unsatisfactory because of toxicity and drug resistance issues. Historically, antifungal screening programs have centered mainly on Candida and Aspergillus spp. There is still a need for treatments effective against these pathogens, but Cryptococcus spp. have been relatively neglected in such programs.3 Given that natural products from fungi have been important in the development of antifungal agents, we have initiated a Cryptococcus-centric exploration of fungi for antifungal natural products, with an emphasis on fungal sources that remain relatively unexplored to date. Coprophilous fungi are those that colonize animal dung and constitute an especially conspicuous component of the microbiota of herbivore dung.4−6 Past studies of coprophilous fungi have afforded a variety of new metabolites with bioactivities that often included antifungal effects.7,8 Even so, this ecological niche group remains relatively underexplored. During our continuing studies of coprophilous fungi, a strain, © XXXX American Chemical Society and American Society of Pharmacognosy

TTI-0426, was isolated from horse dung collected at Lake Houston in Montgomery County, Texas. Morphological characteristics and rDNA sequence analysis identified TTI0426 as an undescribed Niesslia sp. The rDNA sequences were most similar to metagenome sequences amplified from cotton field soil in northern Texas and soil-borne strains of Monocillium indicum. An extract of a solid-substrate fermentation of TTI-0426 exhibited activity against Cryptococcus neoformans and Candida albicans and was therefore selected for chemical investigation. Fractionation of this extract led to the isolation of six new wortmannin and wortmannine derivatives (1−6), which we named wortmannins C and D and wortmannines B1−B4. Special Issue: Special Issue in Honor of Drs. Rachel Mata and Barbara Timmermann Received: November 2, 2018

A

DOI: 10.1021/acs.jnatprod.8b00923 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. 1H and 13C NMR Spectroscopic Data for Wortmannins C (1) and D (2)

RESULTS AND DISCUSSION Strain TTI-0426 displayed morphological features reminiscent of several fungi in the Niessliaceae, especially species of Monocillium and Niesslia. Similarity searches of public databases (NCBI, Mycobank) with the large subunit (LSU) rDNA sequences retrieved Niesslia exilis (AY489720, 96% identity) as the most similar sequence, and searches with the internal transcribed spacer region (ITS) of the rDNA retrieved Canadian soil-borne strains identified as Monocillium indicum (e.g., CBS 182.65, 97% identity) and a series of metagenomic ITS partial sequences from cotton fields near Lubbock, Texas (e.g., JX319019, JX366671, JX321580, 99% identity). Maximum likelihood analysis of the LSU rDNA placed TTI-0426 close to strains of M. indicum (85% branch support). Maximum likelihood analysis of the ITS rDNA also indicated that TTI0426 was closely related to M. indicum or possibly even a sister species. Furthermore, TTI-0426 grouped close with partial ITS metagenomic sequences amplified from soils of northern Texas cotton fields, indicating that it might be the same species. Therefore, we concluded that TTI-0426 represents a yet unnamed species of Niesslia. Methyl ethyl ketone (MEK) extracts of TTI-0426 grown on a solid vermiculite matrix9 infused with sucrose−yeast extract medium (YES)10 or on an agitated liquid sucrose−tomato paste medium (STP)11 caused growth inhibition zones against C. albicans and C. neoformans, but did not affect Staphylococcus aureus (Figure S26). The fermentation was scaled up on YES medium infused into vermiculite. The solid mass was extracted with MEK and concentrated under vacuum. Solvent partitioning, followed by silica gel chromatography and reversed-phase HPLC, afforded compounds 1−6.

1a δH

position 1 2a 2b 3 4 5 6 7 8 9 10 11 12α 12β 13 14 15α 15β 16a 16b 17 18 19 20 21 22 23 24a 24b 25 26

4.76, dd (2.1, 6.9) 3.00, dd (6.9, 11.1) 3.43, dd (2.1, 11.1)

6.17, ddd (2.8, 7.5, 9.0) 1.55, m 2.54, m 2.88, 3.13, 2.04, 2.23, 2.56,

ddd (2.8, 5.9, 12.8) m tt (9.1, 12.8) td (9.1, 18.6) m

0.95, 1.74, 8.22, 3.14,

s s s s

2.43, 1.55, 1.68, 1.18, 0.89,

m m m d (6.8) t (7.4)

2b δC

δH

c

88.4 72.8 157.7 114.3 143.0 144.8 172.7 140.3 149.7 40.8 70.2 36.1 49.3 44.2 23.2 35.8 216.4 14.7 26.5 150.2 59.5 175.6 40.8 27.2 15.8 11.5

4.76, dd (2.1, 6.8) 3.00, dd (6.8, 11.1) 3.43, dd (2.1, 11.1)

6.16, ddd (2.7, 7.5, 8.8) 1.58, dd (8.8, 12.7) 2.70, m 2.88, 3.18, 2.05, 2.23, 2.70,

ddd (2.7, 6.0, 12.8) m tt (9.2, 12.8) td (9.2, 18.8) m

0.95, 1.71, 8.22, 3.13,

s s s s

2.70, m 1.19, d (7.1) 1.20, d (6.8)

a Recorded in CDCl3, at 600, 150 MHz. bRecorded in CDCl3 at 500 MHz. cAll 13C NMR multiplicities are consistent with the assignments.

Wortmannin C (1) was obtained as a yellow oil. The molecular formula was determined to be C26H30O8 (12 degrees of unsaturation) based on HRESIMS data. The 1H and 13C NMR data of 1 (Table 1) revealed the presence of three methyl singlets (one methoxy), one methyl doublet, and one methyl triplet. Resonances for five methylene units, five methines (two oxygenated and one aromatic/olefinic), and 11 nonprotonated carbons were also observed. These data closely resembled those of the known compound wortmannin (7),12 with the only major differences being the absence of a resonance for an acetyl methyl group and the presence of additional signals for two methyl groups (a doublet and a triplet), a methylene unit, and a methine. These differences suggested that the acetyl group in wortmannin is replaced with a 2-methylbutyryl group in 1. This hypothesis was supported by analysis of 2D NMR data. HMBC correlations shown in Figure 1 indicated the planar connectivity of carbons C-1 through C-21 to be the same as that of wortmannin. No correlations to cross-conjugated carbonyl carbon C-7 were

Figure 1. COSY and key HMBC correlations for 1 (CDCl3) and 4 (acetone-d6).

observed, but a signal at δC 172.7 for the corresponding carbon was present in the 13C NMR spectrum, matching well with literature data.12 Correlations from both H3-26 and H3-25 to a methylene carbon at δC 27.2 and a methine carbon at δC 40.8, together with additional correlations from H3-24 and H-23 to ester carbon C-22, completed the 2-methylbutyrate unit. Finally, a correlation from oxymethine H-11 to C-22 confirmed the placement of the 2-methylbutyrate unit at C11, leading to the assignment of structure 1. NOESY correlations (Figure 2) from H-1 to H3-19 and H11, as well as from H-11 to H3-18 and Hβ-12, indicated that B

DOI: 10.1021/acs.jnatprod.8b00923 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 2. Key NOESY correlations for 1 (CDCl3) and 4 (acetone-d6).

Table 2. Selected NMR Spectroscopic Data for Wortmannines B1−B4 (3−6) 3a δH

position

b

1 2a

4.43, d (2.7) 3.27, d (10.5)

2b

3.61, dd (2.7, 10.5)

3 4 5 6 7 8 9 10 11 12α 12β 13 14 15α 15β 16α 16β 17 18 19 20 21 22 23 24a 24b 25 26 NH OH

4.00, d (6.4)

6.00, ddd (3.4, 7.2, 9.7) 1.47, m

δC

85.4 72.1

2.58, m 1.89, tt (8.9, 12.5) 2.18, dt (8.9, 19.5) 2.58, m 1.13, 1.51, 5.59, 3.08,

s s d (6.4) s

2.33, 1.51, 1.64, 1.14, 0.90, 6.31, 3.42,

m m m d (6.9) t (7.5) s s

d

4.49, d (2.4) 3.42, d (10.6)

4 δC

169.1 46.3 64.3 172.1 199.0 141.8 164.4 45.7 68.7 36.1

3.77, d (6.2)

5.96, ddd (3.5, 7.2, 9.9) 1.64, m

85.8 73.4

168.0 47.3 65.5 173.5 200.7 142.7 164.6 46.2 69.7 36.6

2.54, m 50.5 42.3

51.0 42.4

20.0

2.94, ddd (3.5, 5.7, 12.9) 2.51, m 1.83, tt (9.0, 12.9)

20.8

35.5

2.16, m

35.4

2.59, m 215.4 13.9 17.0 77.0 58.2 174.4 41.2 26.9 16.5 11.6

1.09, 1.52, 5.57, 3.05,

s s d (6.2) s

2.57, 1.53, 1.67, 1.16, 0.92, 8.04,

m m m d (6.8) t (7.4) s

δHf

e ,g

3.60, dd (2.4, 10.6)

2.75, dd (7.2, 12.7) 2.58, m

δH

c,g

215.3 14.2 17.4 77.8 58.0 175.6 41.5 27.6 16.5 11.6

4.51, d (2.5) 3.37, d (10.7)

δH

d

3.62, dd (2.5, 10.7)

4.54, t (2.7) 3.52, dd (2.7, 11.4) 3.74, dd (2.7, 11.4)

3.83, d (6.6)

3.64, d (1.2)

5.99, ddd (3.5, 7.0, 10.0) 1.59, m

5.99, ddd (3.4, 7.0, 10.0) 1.64, m

2.56, m

2.54, m

2.88, dd (3.5, 6.0, 12.9) 2.49, m 1.80, tt (9.0, 12.9)

2.99, ddd (3.4, 5.9, 12.9) 2.51, m 1.85, tt (9.0, 12.9)

2.24, dt (9.0, 18.8) 2.56, m

2.18, dt (9.0, 19.1) 2.59, m

1.10, 1.53, 5.48, 3.08,

s s d (6.6) s

1.06, 1.39, 5.52, 3.16,

s s s s

2.56, 1.59, 1.66, 1.18, 0.95,

m m m d (6.9) t (7.5)

2.57 1.55 1.67 1.15 0.92 8.17

(m) (m) (m) (d, 6.9) (t, 7.4) (s)

δCe,g 85.4 73.2

169.2 50.7 66.5 170.7 202.3 142.9 167.2 47.3 69.2 36.4

50.9 42.2

5

6

δHb

δH b

4.46, d (2.6) 3.30, d (10.5) 3.63, dd (2.6, 10.5)

4.42, t (2.6) 3.44, dd (2.6, 11.4) 3.76, dd (2.6, 11.4)

4.03, d (6.4)

3.80, s

6.02, ddd (3.2, 7.2, 10.0) 1.42, m 2.75, dd (7.2, 12.6)

6.04, ddd (3.4, 7.0, 10.0) 1.48, dd (10.0, 12.7) 2.74, dd (7.0, 12.7)

2.55, m

2.59, m

20.9

2.55, m 2.59, m 1.89, tt (8.9, 12.2) 1.92, tt (9.1, 13.1)

36.0

2.19, dt (8.9, 18.4) 2.55, m

2.24, dt (9.1, 19.4) 2.59, m

1.11, 1.48, 5.62, 3.10,

1.08, 1.44, 5.65, 3.18,

215.0 14.1 17.7 80.3 58.2 175.5 41.4 27.6 16.4 11.6

s s d (6.4) s

s s s s

2.55, m 1.163, d (7.0)

2.59, m 1.19, d (7.0)

1.158, d (7.0)

1.19, d (7.0)

6.22, s

6.25, s

a

Data for 3 are given in three different solvents to foster comparison with literature analogues as well as 4−6. bMeasured in CDCl3 at 500 MHz. Measured in CDCl3 at 150 MHz. dMeasured in acetone-d6 at 600 MHz. eMeasured in acetone-d6 at 125 MHz. fMeasured in methanol-d4 at 500 MHz. gAll 13C NMR multiplicities are consistent with the assignments. c

C

DOI: 10.1021/acs.jnatprod.8b00923 J. Nat. Prod. XXXX, XXX, XXX−XXX

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configuration, matching that of wortmannine B and wortmannin at analogous stereocenters. Wortmannine B2 (4) was determined to be an isomer of 3. The 1H NMR spectrum of 4 was very similar to that of 3 with the main differences being the shift of H-4 from δ 4.03 in 3 to 3.80 in 4 and a difference in JH4−H20 from 6.5 Hz in 3 to ∼0 in 4, suggesting that 4 is an epimer of 3, likely differing only in the configuration at C-20. COSY and HMBC data for 4 confirmed the same planar structure as 3, and NOESY data indicated the same relative configuration at all stereocenters except for C-20. Energy-minimized models of each epimer, generated using Spartan 16, were utilized to measure the dihedral angle between H-4 and H-20. The 6.5-Hz coupling constant in 3 is consistent with a predicted dihedral angle of 41°, closely matching the data for wortmannine B and indicating that H-20 is α-oriented. Conversely, the value in 4 is near zero, consistent with a predicted dihedral angle closer to 90°, indicating that H-20 is β-oriented in 4. These data establish 4 as an epimer of 3 at C-5, leading to assignment of the absolute configuration shown. Compound 5 was assigned a molecular formula containing one CH2 unit less than 3 and 4 on the basis of HRESIMS data. The 1H NMR spectrum of 5 was very similar to that of 3, with the signals for the 2-methylpropyl unit replaced by those of an isopropyl group, indicating that the 2-methylbutyryl unit in 3 was replaced by an isobutyryl unit in 5. HRESIMS data for 6 showed it to be an isomer of 5. NMR data revealed that the only significant difference between 5 and 6 was analogous to the difference between 3 and 4, indicating that 6 is an epimer of 5 at position C-20. The names wortmannines B3 and B4 are proposed for 5 and 6, respectively. Compounds 1−6 were evaluated for activity against C. neoformans, C. albicans, and S. aureus in disk diffusion assays at levels of 50 μg/disk. Compounds 1 and 2 showed 16 and 13 mm inhibitory zones, respectively, against C. neoformans (amphotericin B gave a 13 mm zone at 25 μg/disk). Both compounds also showed 25 mm zones at 50 μg/disk against C. albicans (amphotericin B gave a 10 mm zone at 25 μg/disk), but did not show activity against S. aureus. Compounds 3−6 were inactive in all of these assays. To better define the antifungal effect of representative compound 1, minimal inhibitory concentration (MIC) values were determined for 1 by the broth microdilution method according to NCCLS protocols, testing against Aspergillus f umigatus, as well as C. albicans and C. neoformans. Disappointingly, at 48 h, the MIC values for C. albicans and A. f umigatus were each 200 μM, while the MIC for C. neoformans (as measured at 72 h) was 100 μM (ca. 47 μg/mL). Interestingly, visual inspection of growth at 24 h indicated a significant growth delay of the C. albicans cultures at drug concentrations as low as 25 μM. However, inhibition at lower concentrations was temporary, with growth similar to untreated controls at the 48 h end-point at concentrations below the formal MIC. This observation suggests an initial fungistatic effect that is eventually overcome with longer incubation times. To further explore the potential mechanism of this temporal growth inhibition, we performed MIC testing for 1 in the Schizosaccharomyces pombe isolate MJ1682, a yeast strain with mutations in several efflux pump genes, as well as a mutation in the gene encoding a transcription factor regulating drug efflux. This strain has been used to assess the role of MDR-type efflux pumps in antifungal drug resistance and fungal cell physiology.16,17 Compared to the isogenic wild-type control

these are all on the same face of the ring system, while a correlation from Hα-12 to H-14 placed these protons on the opposite face. These correlations were fully consistent with a relative configuration analogous to that of 7. The absolute configuration of 1 is assumed to match that of 7 at the relevant stereocenters. Though not conclusive, the specific rotation of 1 is similar to that of 7 (1: [α]20D = +72; 7: [α]20D = +86.5). Unfortunately, small-scale hydrolysis of a sample of 1 failed to afford a 2-methylbutyric acid degradation product for comparison to standards, so the absolute configuration at the corresponding stereocenter was not assigned. The HRESIMS data for wortmannin D (2) indicated a molecular formula with one less CH2 unit than 1. The 1H NMR spectrum again resembled that of wortmannin. In comparison to 1, 2 lacked signals for the 2-methylbutyryl unit, instead showing signals for an isopropyl group. These data suggested that the 2-methylbutyryl unit in 1 is replaced by an isobutyryl unit in 2, and all NMR data for 2 (Table 1) were fully consistent with this assignment, as all other spectroscopic data were nearly identical to those of 1. It is assumed that the stereochemical configuration of 2 is analogous to that of wortmannin and 1. The 2-methylbutyryl and isobutyryl units are somewhat rare as constituents of fungal metabolites Wortmannin (7) is a well-known potent phosphoinositide-3kinase inhibitor originally described from Penicillium wortmanni as a selective antifungal agent13 and subsequently reported from several other fungal sources. Multiple analogues have been prepared synthetically in efforts to optimize its kinase inhibitory effects, but only a few analogues have been isolated from other fungal sources, including wortmannin B, which lacks the C-2 methoxy methyl group, while replacing the acetyl group of 7 with a methyl group.14 Given the prior nomenclature, the names wortmannin C and D are proposed for 1 and 2. The NMR data for wortmannine B1 (3) resembled those of 1 and 2, but 3 was found to incorporate a nitrogen atom, as it was assigned the molecular formula C26H33NO9 (11 degrees of unsaturation) on the basis of HRESIMS data. The 1H and 13C NMR data (Table 2) again showed resonances for a 2methylbutyryl unit, along with three methyl singlets (including one methoxy), one oxygenated methylene, three other aliphatic methylene units, five other methines (including three oxygenated), and nine other nonprotonated carbons (two olefinic, two other ester/amide carbons, two ketones, and three quaternary sp3 carbons). These data closely matched those of the recently reported wortmannine B,15 which contains a previously undescribed ring system and whose structure was confirmed by X-ray crystallographic analysis. The NMR data suggested that the structure of 3 matches that of wortmannine B with acylation of the C-11 oxygen by a 2-methylbutyryl unit. Wortmannine B lacks an acyl unit altogether at that position. COSY and HMBC (Figure 1) correlations for 3, including HMBC correlations from H-23 and H-11 to C-22, independently confirmed this hypothesis. NOESY correlations (Figure 2) from H-11 to Hβ-12, H3-18, and H3-19, as well as from H3-19 to H-1, placed all of these hydrogens on the same face of the molecule, while correlations from Hα-12 to H-14 and from H3-21 to H-4 placed these units on the opposite face. The coupling constant observed between H-4 and H-20 matched well with that reported for wortmannine B, indicating the same relative configuration at C-5 and establishing an overall relative configuration, and presumably absolute D

DOI: 10.1021/acs.jnatprod.8b00923 J. Nat. Prod. XXXX, XXX, XXX−XXX

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°C, and extension for 2 min at 72 °C, with final extension at 72 °C for 10 min. Amplicons were sequenced using the primers ITS1, ITS4 (5′TCCTCCGCTTATTGATATGC-3′), LROR (5′-ACCCGCTGAACTTAAGC-3′), LR5 (5′-TCCTGAGGGAAACTTCG-3′), and LR7.20,21 The ITS+LSU rDNA sequence (1921 bp) was accessioned in the NCBI GenBank (MH997627). The sequence of the LSU region of TTI-0426 was aligned with sequences from other species of Monocillium, Niesslia, and Hyaloseta retrieved from GenBank. These three genera recently have been merged into the genus Niesslia.22 A second alignment was made using the ITS region and similar sequences from GenBank. Maximum likelihood analyses of the two sets of aligned sequences were executed in Mega 7.0,23 producing phylogenetic trees (Figures S29 and S30). Based on the phylogenies of its LSU and ITS sequences, TTI-0426 appears to be a possible sister species of M. indicum, the type species of the genus Monocillium.22,24 The close relationship to M. indicum was also evident in the ITS phylogeny. However, unlike M. indicum, the conidiogenous cells of TTI-0426 are aggregated in sporodochial masses and the conidia are allantoid in shape. TTI-0426 was even more closely related to, and possibly conspecfic with, a group of metagenomic sequences amplified from northern Texas cotton fields. Therefore, we refer to TTI-0426 as Niesslia sp. based on its morphological features and its phylogenetic proximity to other species of Niesslia and Monocillium. Fermentation and Extraction. The strategy to generate smallscale extracts from fungi for antifungal screening against C. neoformans and C. albicans has been detailed previously.25,26 Briefly, the strain was grown at a 12 mL scale on five media selected for proven ability to stimulate production of secondary metabolites. After 14 d, each fermentation was extracted with an equal volume of MEK. After thorough mixing of the solvent with the culture for at least 2 h, 5 mL of the organic phase was dried. The dried residue was resuspended in 500 μL of DMSO. Aliquots (20 μL) of the crude DMSO extracts were applied to the wells of the assay plates as described above along with the corresponding controls, e.g., amphotericin B or a mixture of streptomycin sulfate and chlortetracycline (Figure S26). To scale up the growth of TTI-0426, agar plugs from YMA cultures were inoculated into 250 mL Erlenmeyer flasks each containing 50 mL of SMYA seed medium (1% Bacto neopeptone, 4% maltose, 1% yeast extract, and 0.4% agar). Seed media were incubated for 5 days with agitation at 220 rpm at 23 °C. A scaled-up fermentation was then carried out in eight 1 L Pyrex bottles each containing 340 mL of coarse sterilized vermiculite. Liquid YES medium10 consisting of 150 g sucrose; 20 g yeast extract; 0.5 g MgSO4·7H2O; 1 mg ZnSO4·7H2O; and 0.5 mg CuSO4·0.5H2O in 1 L of deionized H2O was prepared in 125 mL aliquots in separate flasks. A 5 mL aliquot of seed culture was mixed into each flask, and the mixture from each flask was poured into a bottle of the sterilized vermiculite. The bottles were incubated and rolled on a tissue culture roller machine at 23 °C until the fungal growth penetrated and immobilized the vermiculite (4 d). After 14 d, the growth was extracted with an equal volume of MEK by rolling the bottles with the solid matrix for 2 h. Isolation and Purification. The crude extract (980 mg) was partitioned between hexane (3 × 15 mL) and MeCN (20 mL). The MeCN-soluble portion (858 mg) was subjected to silica gel column chromatography, eluting with a stepwise gradient of hexane−EtOAc (200 mL each of 1:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 0:1) and EtOAc−MeOH (100 mL of 99:1, 97:3, 95:5, 85:15, 75:25, 50:50, 25:75, 0:100, 0:100) to provide 20 fractions. Fraction 7 (25 of 27 mg) was purified by reversed-phase HPLC with 65% MeCN−H2O for 15 min, followed by a linear gradient to 100% MeCN−H2O over 15 min to afford 2 (1.3 mg, tR = 14.6 min) and 1 (5.4 mg, tR = 17.5 min). Fraction 9 (37 of 48 mg) was separated using reversed-phase HPLC with 40% MeCN−H2O for 20 min, followed by a linear gradient to 100% MeCN−H2O over 10 min to give 6 (1.6 mg, tR = 21.5 min) and 4 (4.6 mg, tR = 28.1 min). Fraction 10 (48 of 51 mg) was purified using reversed-phase HPLC with 20% MeCN−H2O for 20 min, followed by a linear gradient to 100% MeCN−H2O over 10 min to afford 5 (3.2 mg, tR = 18.3 min), 6 (1.9 mg, tR = 21.6 min), 3 (5.4 mg, tR = 25.9 min), and 4 (3.0 mg, tR = 28.2 min). Fraction 11 (28 of 32

strain SAK1, the MJ1982 efflux mutant was 10 times more susceptible to compound 1. Taken together, these observations suggest that efflux from the fungal cell might limit the potency and durability of the antifungal activity of this compound. Wortmannin (7) and related compounds such as viridin and its analogues are known to be derived from the steroid pathway via distinctive steps that involve oxidative removal of carbons from sterol precursors.18 The recent publication of the viridin biosynthetic gene cluster18 helped guide the identification of a putative gene cluster in a draft genome sequence of strain TTI0426 that could potentially encode the biosynthesis of wortmannin and wortmannine analogues (Supporting Information, Figures S31−S33 and Tables S1−S3). Members of this class that contain the furan unit characteristic of 1, 2, and 7 are referred to as furanosteroids, and compounds retaining this unit are well known to display a variety of bioactivities. The mechanism leading to the skeletal change in 3−6 or the stage of the biosynthetic process at which it occurs is not clear, but the absence of this unit in 3−6 is consistent with loss of activity among these analogues.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Rudolph Research Autopol III automatic polarimeter. UV data were obtained using a Varian Cary III UV/vis spectrophotometer. 1H and 13C NMR spectra were recorded using an AVANCE-500 spectrometer with a 5 mm probe or an AVANCE600 spectrometer with a 1.3 mm probe. Chemical shift values were referenced to residual solvent signals for acetone-d6 (δH/δC, 2.05/ 29.8), CDCl3 (δH/δC, 7.24/77.2), or methanol-d4 (δH/δC, 3.31/49.0). HSQC, HMBC, COSY, and NOESY data were recorded using an AVANCE-600 spectrometer with a 1.3 mm probe or a Bruker AVANCE-500 MHz spectrometer with a 5 mm triple-resonance cryoprobe. HRESIMS data were recorded using a Waters Q-TOF Premier mass spectrometer. Reversed-phase HPLC separations were carried out using a semipreparative Apollo C18 column (Alltech; 1.0 × 25 cm, 5 μm particles; 2 mL/min) interfaced with an Agilent 1220 Infinity instrument equipped with a variable-wavelength UV detector or a Gilson model 322 instrument equipped with a model 155 variable-wavelength detector. Fungal Material. Horse dung was collected at Lake Houston Wilderness Park, Montgomery Co., Texas, USA. The producing strain was isolated by plating washed dung particles onto Miru’s agar medium.19 Mycelia emerging from these dung particles were transferred to YMA medium (1% maltose extract, 0.2% yeast extract, and 2% agar) to establish axenic cultures. The isolate was assigned the accession number TTI-0426 in Texas Therapeutics Institute’s culture collection at the Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at Houston, Houston, TX, USA. Strain TTI-0426 grew on agar media to form slow-growing white mycelium bearing pink, slimy, sporodochial conidiomata. Thick-walled lanceolate and capitate setae formed among conidiogenous cells. The phialidic conidiogenous cells produce slimy masses of curved elliptical to allantoid conidia. This combination of features was reminiscent of several fungi in the Niessliaceae, especially species of Monocillium and Niesslia with thick-walled conidiogenous cells and capitate setae. Key morphological features that identify the fungus as a species of Niesslia are illustrated in Figures S27 and S28. To corroborate the morphological identification, we estimated its approximate phylogenetic relationships based on the ITS and LSU rDNA sequences. DNA extraction from cultured mycelium was conducted as previously described. The ITS and LSU DNA sequences were amplified by primer ITS1 (5′-TCCGTAGGTGAACCTGCGG3′) and LR7 (5′-TACTACCACCAAGATCT-3′).9 DNA was amplified in 50 μL reaction volumes by the following procedures: initial denaturation at 94 °C for 2 min, followed by 32 cycles consisting of denaturation at 94 °C for 30 s, annealing for 30 s at 55 E

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determined at 48 h for C. albicans and A. f umigatus and 72 h for C. neoformans due to its slower growth rate. Due to nutritional auxotrophies, the S. pombe strains were incubated in YES medium for 72 h.28 Viability was determined with the aid of a plate reader using Alamar Blue (BioRad) as the viability indicator. The strains employed were efflux mutant MJ1682 (h90 ade6 leu1 ura4-D18 caf5::bsdR pap1-del pmd1-del mfs1-del bfr1-del) and wild-type control SAK1(h+ ade6-M210 leu1 ura4-D18). Itraconazole was used as a positive control.

mg) was separated using reversed-phase HPLC with 40% MeCN− H2O for 20 min, followed by a linear gradient to 100% MeCN−H2O over 10 min to give 5 (2.4 mg, tR = 18.1 min) and 3 (3.3 mg, tR = 25.7 min). Wortmannin C (1): yellow oil; [α]25D +72 (c 0.003, MeOH); UV (MeOH) λmax (log ε) 205 (4.0), 256 (3.8), 289 (3.7) nm; 1H and 13C NMR data, see Table 1; HMBC data, H-1 → C-2, 3, 5, 10, 19; Ha-2 → C-1, 21; Hb-2 → C-10; H-11 → C-8, 9, 10, 12, 22; Hα-12 → C-11, 13, 14, 17, 18; Hβ-12 → C-9, 11, 13, 14, 17, 18; H-14 → C-8, 9, 13, 15, 16, 18; Hα-15 → C-13, 14, 17; Hβ-15 → C-8, 14, 16; Ha-16 → C15, 17; Hb-16 → C-14, 17; H3-18 → C-12, 13, 14, 17; H3-19 → C-1, 5, 9, 10; H-20 → C-4, 5, 6; H3-21 → C-2; H-23 → C-22, 24, 25, 26; Ha-24 → C-22, 23, 25, 26; Hb-24 → C-22, 23, 25, 26; H3-25 → C-22, 23, 24; H3-26 → C-23, 24; key NOESY data, H-1 ↔ H3-19; H-1 ↔ H-11; H-11 ↔ H3-18; Hβ-12; Hα-12 ↔H-14; H-14 ↔ Hα-15; HRESITOFMS m/z 493.1833 [M + Na]+ (calcd for C26H30O8Na, 493.1838). Wortmannin D (2): yellow oil; [α]25D +30 (c 0.00087, MeOH); UV (MeOH) λmax (log ε) 204 (3.6), 256 (3.3), 284 (2.7) nm; 1H NMR data, see Table 1; HRESITOFMS m/z 479.1681 [M + Na]+ (calcd for C25H28O8Na. 479.1682). Wortmannine B1 (3): white powder; [α]25D +40 (c 0.0033, MeOH); UV (MeOH) λmax (log ε) 203 (3.6), 249 (3.6) nm; 1H and 13 C NMR data, see Table 2; HMBC data, NH → C-4, 5, 6, 20; H-1 → C-3, 5, 10, 19; Ha-2 → C-1, 10, 21; Hb-2 → C-1, 10, 21; H-4 → C-3, 5, 7, 10, 20; H-11 → C-8, 9, 12, 22; Hα-12 → C-11, 13, 14, 18; Hβ-12 → C-9, 11, 13, 14, 18; H-14 → overlap; Hα-15 → C-8, 14, 16; Hβ-15 → overlap; Hα-16 → C-14, 15, 17; Hβ-16 → overlap; H3-18 → C-12, 13, 14, 17; H3-19 → C-1, 5, 9, 10; H-20 → C-5, 6; H3-21 → C-2; H23 → C-22, 24, 25, 26; Ha-24 → C-22, 23, 25, 26; Hb-24 → C-22, 23, 25, 26; H3-25 → C-22, 23, 24; H3-26 → C-23, 24; key NOESY data, H-1 ↔ H3-19; H3-19 ↔ H-11; H-11 ↔ H3-18; H-11 ↔ Hβ-12; Hα-12 ↔ H-14; H3-18 ↔ Hβ-15; H-4 ↔ H3-21; HRESITOFMS m/z 526.2054 [M + Na]+ (calcd for C26H33NO9, 526.2053). Wortmannine B2 (4): white powder; [α]25D +51 (c 0.0032, MeOH); UV (MeOH) λmax (log ε) 204 (3.8), 246 (3.8) nm; 1H and 13 C NMR data, see Table 2; HMBC data, NH → C-4, 5; H-1 → C-3, 5, 10, 19; Ha-2 → C-1, 10, 21; Hb-2 → C-1, 10, 21; H-4 → C-3, 5, 6, 7, 20; H-11 → C-8, 9, 12, 22; Hα-12 → C-11, 13, 18; Hβ-12 → C-9, 11, 13, 14, 17, 18; H-14 → C-8, 9, 12, 13, 15, 18; Hα-15 → C-13, 14, 16, 17 ; Hβ-15 → C-8, 14, 16; Hα-16 → C-15, 17; Hβ-16 → C-14, 15, 17; H3-18 → C-12, 13, 14, 17; H3-19 → C-1, 5, 9, 10; H-20 → C-3, 5, 6; H3-21 → C-2; H-23 → C-22, 24, 25, 26; Ha-24 → C-22, 23, 25, 26; Hb-24 → C-22, 23, 25, 26; H3-25 → C-22, 23, 24; H3-26 → C-23, 24; key NOESY data, H-1 ↔ H3-19; H3-19 ↔ H-11; H-11 ↔ H3-18; H318 ↔ Hβ-15; Hα-15 ↔ H-14; H-14 ↔ Hα-12; H-14 ↔ Hα-16; H-4 ↔ H3-21; H3-21 ↔ H-14; HRESITOFMS m/z 526.2060 [M + Na]+ (calcd for C26H33NO9Na, 526.2053). Wortmannine B3 (5): white powder; [α]25D +42 (c 0.0013, MeOH); UV (MeOH) λmax (log ε) 203 (3.6), 248 (3.5) nm; 1H NMR data, see Table 2; HRESITOFMS m/z 512.1898 [M + Na]+ (calcd for C25H31NO9Na, 512.1897). Wortmannine B4 (6): white powder; [α]25D +20 (c 0.0011, MeOH); UV (MeOH) λmax (log ε) 204 (3.4), 243 (3.2) nm; 1H NMR data, see Table 1; HRESITOFMS m/z 512.1898 [M + Na]+ (calcd for C25H31NO9Na, 512.1897). Antifungal Assays. In vitro antifungal activity was measured according to methods recommended by the National Committee for Clinical Laboratory Standards (NCCLS).27 The strains and methods for agar zone of inhibition assays with C. albicans ATCC 10231, C. neoformans H99, and S. aureus ATCC 43330 have been detailed previously.22 MIC values were also measured according to NCCLS recommendations, using the serial broth microdilution method in 96well plates. The test medium was RPMI-1640 (Sigma) supplemented with 0.165 M MOPS, pH 7.0. Compounds were dissolved in DMSO and serially diluted in the medium. The spectrophotometric MIC value was defined as the lowest concentration of a test compound that resulted in a culture with a density consistent with 50% growth inhibition (MIC-50) when compared to the growth of the untreated control. The strains were incubated at 37 °C, and the MICs were

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00923. 1 H NMR, 13C NMR, and 2D NMR spectra for compounds 1 and 4, 1H NMR, DEPT, and 2D NMR spectra for compound 3, and 1H NMR spectra for compounds 2, 5, and 6 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Gerald F. Bills: 0000-0003-2352-8417 James B. Gloer: 0000-0002-9261-7571 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from NIH (R01 GM121458), the Chinese Scholarship Council (to L.X.), and the Kay and Ben Fortson Endowment (to G.B.). We thank W. Gams and J. B. Stielow of the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands, for allowing us access to their unpublished revision of the genus Niesslia. Financial support for the 600 MHz NMR and HRMS instruments employed in this work was provided by grants from NIH (S10 RR025500) and NSF (CHE-0946779), respectively. Genome sequencing was supported by the National Natural Science Foundation of China (No. 31870528). Technical assistance from the staff of the University of Iowa NMR and MS facilities is greatly appreciated.

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DEDICATION Dedicated to Dr. Rachel Mata, National Autonomous University of Mexico, Mexico City, Mexico, and Dr. Barbara N. Timmermann, University of Kansas, for their pioneering work on bioactive natural products.

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REFERENCES

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